Hyperoxia Causes Inducible Nitric Oxide Synthase-Mediated Cellular Damage to the Immature Rat Brain

Abstract

Relative hyperoxia is a condition frequently encountered in premature infants, either spontaneously or during treatment in the Neonatal Intensive Care Unit. The effects of high inspiratory oxygen concentrations on immature brain cells and their signaling cascades are largely unknown. The aim of the study was to investigate the effect of hyperoxia on the amount and topographic distribution of iNOS-expression (inducible nitric oxide synthase) in the immature rat brain, and to localize hyperoxia-induced formation of peroxynitrite as a potential marker of cellular damage to immature cerebral structures. Seven-day-old Wistar rat pups were exposed to Ͼ80% oxygen for 24 h and were then transcardially perfused. Following paraformaldehyde fixation, brains were paraffin-embedded and immunohistochemically stained for iNOS and nitrotyrosine. iNOS protein was quantified by Western blot; iNOS mRNA expression was studied by RT-PCR. Total brain iNOS mRNA was up-regulated, demonstrating a peak at 6 h following the onset of hyperoxia. Immunohistochemical staining was predominantly observed in microglial cells of hippocampus and frontal cortex with some iNOS reactivity in endothelial and perivascular cells. Nitrotyrosine staining was positive in apical dendrites of neurons in the frontal cortex. The role of NO has been subject of numerous investigations during recent years. This applies to its function as a gaseous messenger molecule and to its vasodilative properties. NO is released by hydroxylation of nitrogen at the guanidino group of L-arginine, a reaction which is catalyzed by the enzyme NO synthase (NOS). At least three isoforms of NOS are known at the moment, two constitutive forms (endothelial NOS and neuronal NOS) and the inducible form (iNOS). The latter is induced by bacteria and cytokines. NO concentrations produced by iNOS are much higher than those generated by constitutive NOS (1), and have been shown to exert antimicrobial effects against some bacterial pathogens in vitro (2).As soon as NO is generated in the target cell (cancer cell, parasite), binding at copper and iron containing proteins takes place (3). As a result copper and iron are released and, in combination with oxygen, toxic radicals are formed. This is referred to as "massive oxidative injury" and requires the expression of iNOS leading to the production of huge amounts of NO, whereas low levels of NO synthesized by the constitutive forms of NOS (eNOS, nNOS) act as a signal molecule (4). Thus, the concentration of NO determines its biologic effect upon neighboring cells and tissues. The reaction products of nitrogen and oxygen include the strong oxidant peroxyni-

Postischemic hyperoxia reduces hippocampal pyruvate dehydrogenase activity

“…Whereas prolonged hyperoxia can stimulate the expression of inducible nitric oxide synthase [37], increased levels of this protein are not likely to occur within the 2-h reperfusion period used in our measurements. Elevated brain tissue O 2 tension could, however, directly increase the rate of existing redox protein-mediated production of both nitric oxide and superoxide.…”

“…Although reports indicate that severe hypoxia can also stimulate superoxide production [40][41][42], promotion of superoxide generation by high brain tissue pO 2 is a more likely explanation for the increased 3-nitrotyrosine immunoreactivity in the hyperoxic-resuscitated animals. In addition, the K m of O 2 for nitric oxide synthases has been reported to be as low as micromolar or as high as millimolar concentrations, suggesting that elevated tissue O 2 tension could also directly stimulate nitric oxide production [37,43,44]. As microdialysis measurements indicate that total end products of nitric oxide generation are elevated within 20-60 min of reperfusion after transient global cerebral ischemia [45], it follows that hyperoxic resuscitation should increase markers of peroxynitrite production, e.g., 3-nitrotyrosine immunoreactivity.…”

Acute and long-term proteome changes induced by oxidative stress in the developing brain

“…Hyperoxia causes acute functional and morphological changes of the immature brain. [8][9][10] In addition, acute functional and morphological changes may induce further functional and morphological changes in the period of rapid brain development and thereby cause an irreversible damage in the adult brain.…”

“…21,22 Decreased levels of PEA-15 protein have been associated with astrocyte protection against TNF-induced apoptosis. 23 In addition to these differentially regulated proteins that have been linked to apoptosis and oxidative stress and are accompanied by morphological correlates, [8][9][10] a dysregulation of proteins associated with cell growth and energy metabolism suggests that these biological processes are also affected by an exposure to high oxygen levels during the brain growth spurt period (Table 1; Figure 2b). …”

“…However, in many cases, there is neither an obvious clinical explanation nor a corollary on conventional imaging studies for the neuropsychologic morbidity. Recent work has emphasized the role of intrauterine and neonatal infections, 5 pharmacologic agents 6,7 and oxygen [8][9][10] in triggering neuronal death in the developing mammalian brain.…”